WO2022235146A1 - Pharmaceutical composition containing pentacyclic triterpenoids - Google Patents

Abstract

The present invention relates to pharmaceutical compositions containing synergic combinations of pentacyclic triterpenoids as active ingredients and it indicates that certain specific combinations of these drugs have the effect of preventing or inhibiting viral infections. The compositions are found in specific amounts and proportions that improve the pharmacological properties of the compounds, improving their bioavailability, reducing toxicological effects and irritability, especially in the area of the airways and the lungs.

Description

Pharmaceutical composition containing pentacyclic triterpenoids.

Industrial property rights reserved

A part of the description of this application contains material subject to protection of industrial property rights. The owner of such rights has no objection to the reproduction by facsimile of the patent document or application description by any person, as it appears in the patent file or records at the Patent and Trademark Office, but otherwise , reserves all industrial property rights.

FIELD OF THE INVENTION

The present invention deals with pharmaceutical compositions containing pentacyclic triterpenoids as active ingredients, especially glycyrrhizic acid and 18-β-glycyrrhetinic acid. Said compositions are characterized by being in combination with pharmaceutically acceptable excipients and adapted to be administered in humans. Said compositions are suitable for administration by the oral, nasal, cutaneous, intravenous injectable or inhaled routes and for use in the treatment of respiratory diseases, especially viral infectious diseases. The present formulations are especially effective for use in the prevention, treatment and post-treatment of respiratory infections, particularly those whose contagion is through the mechanism of interaction with angiotensin-converting enzyme 2, ACE2, as occurs, among others, with viruses such as severe acute respiratory syndrome coronavirus, SARS-CoV and type 2, SARS-CoV2. It is important to point out that there are various references related to pentacyclic triterpenoids and specifically to the components glycyrrhizic acid, (also designated as AG) and 18-b glycyrrhetinic acid, (also designated as enoxolone or EN). Said references indicate different common names for each of said components; In this writing, the indicated components correspond to the following structures:

Glycyrrhizic acid, (AG) 18-b glycyrrhetinic acid, (EN)

Brief description of the invention

The present invention refers to pharmaceutical compositions that contain combinations of pentacyclic triterpenoids as active ingredients and indicates that some specific combinations between these types of drugs result in surprising effects to prevent or inhibit viral and/or respiratory infections. The compositions are found in specific quantities and proportions that improve the pharmacological properties of the compounds, improving their bioavailability, pharmacokinetics and reducing toxicological and irritability effects, especially in the area of the respiratory tract and lungs.

The compositions described herein are adapted to be administered by intravenous, cutaneous, oral, nasal or inhaled in humans. The compositions can be applied by one or more of these routes, in order to improve the bioavailability of the active ingredients and achieve a high local concentration in the respiratory tract (nasal cavities, pharynx, larynx, trachea, bronchi and bronchioles) and the lungs, where it is especially vulnerable to infection by viral agents.

The different antiviral, antioxidant, antibacterial, analgesic, anti-inflammatory, regenerative and immunomodulatory properties of the active ingredients used in this composition act together to combat respiratory tract infections, reduce the severity of the symptoms associated with them, reduce and relieve post-infection sequelae. The combination of the pharmacological properties of the present invention makes the compositions especially useful for use in the prevention, treatment and recovery of patients suffering or suffering from viral infections, especially those caused by contagion via virus interaction with angiotensin-converting enzyme 2 , (ACE2).

Among the preferred pentacyclic triterpenoids is 18-b glycyrrhetinic acid (EN), which belongs to the bioclassification system II, presenting low polarity, high hydrophobicity and moderate permeability, so it can be mentioned that the limiting step for the Absorption into the bloodstream is the dissolution of the drug, to later be absorbed and distributed to different organs or to the target site.

Another of the preferred pentacyclic triterpenoids is glycyrrhizic acid (AG), of which it can be mentioned that, despite being soluble in water, it has a reduced permeability (belongs to bioclassification system III), therefore, its absorption is a function of the excipients and manufacturing processes.

On the other hand, the selection of excipients and pharmaceutical form define the absorption of the drug and thus the potentiation of the desired therapeutic effect. For the specific case of inhaled pharmaceutical forms, we can mention that these are used when the site of action is focused on the upper airways (nasal cavity, throat, mouth, and larynx) and lower airways (trachea, bronchi, bronchioles, and lungs) and where the pharmaceutical form to be administered does not necessarily need to be absorbed, since the largest proportion of the drug has been put in contact with the site of action. It should be noted that the absorption of the drug will promote a homogeneous distribution in the target organ and in the circulatory system, which leads to the potentiation of the therapeutic effect of the drug to be administered, likewise this absorption can be carried out both in the airways and in the gastrointestinal tract, absorbing the portion that was not absorbed in the upper airways and that is the result of the intake of liquids or food.

The promotion of drug absorption at the site of action enhances the therapeutic effect and is estimated to reduce systemic effects caused by the disease to be treated (collateral damage), for the specific case of EN, in addition to its antiviral effectiveness, its absorption and distribution can promote effects such as: anti-inflammatory, antioxidant, antimicrobial, immunomodulatory, re-epithelializing, antifibrotic, in addition to having the ability to cross the blood-brain barrier. Background

Respiratory tract infections are one of the most frequent diseases that globally affect all population groups. Although the origins of these may be due to various agents, those of viral etiology belong to the technical field of this application. Viruses that are commonly responsible for such diseases belong to the orthomyxoviridae, paramyxoviridae, picornaviridae, coronavirus, and adenovirus families.

Respiratory diseases have an impact in terms of health, quality of life and socioeconomics, so it is important to have treatments and vaccines for their control and prevention. Drugs commonly used to treat viral infections include adamantanes (amantadine and rimantadine), neuraminidase inhibitors (zanamivir and oseltamivir), ribavirin, cidofovir, and pleconaril, among others. The problem with these drugs is that they cause severe side effects, can be very expensive, or can be made obsolete by new viral variants. Other treatments for viral infections include the use of plasma from people convalescing from viral infections, monoclonal antibodies, antisense oligonucleotides, peptides, steroids, and interferons. However, for many of these treatments further studies are still needed to verify their efficacy and safety.

Although in most viral infections, it is enough to administer one of the known drugs, or with the application of palliative treatments; The lack of antiviral drugs is especially critical in the face of the sudden emergence of viral strains with pandemic potential, since in the absence of effective treatments it is difficult to contain epidemic outbreaks. They are cited briefly and as examples, the outbreak of the 1918 H1N1 influenza virus caused 40 to 100 million deaths. Between 2002-2003, a new severe form of pneumonia caused by a coronavirus (SARS-CoV) emerged, leaving a balance of 8,096 cases and 744 deaths in 29 countries on 5 continents. In 2009, a new variant of influenza A-H1N1 caused a pandemic with an estimated 100,000 to 400,000 deaths globally. This denotes that viral epidemics periodically arise with a severe impact that depends on the virulence of the strain with which it is treated.

At the end of 2019, with the sudden emergence of the SARS-CoV2 coronavirus strain, there are records for the year 2020 and during the first months of 2021, of more than 2,500,000 deaths worldwide. With regard to this virus, although 80% of those infected do not progress to a serious clinical picture, the remaining 20% have severe symptoms and between 3 and 10% may have a fatal outcome. In addition to the symptoms present during the course of the infection, it has been observed that, in some cases, those who suffered from the coronavirus infection suffer sequelae such as damage to the tissue of organs such as the heart, lungs, nervous system and brain, damage to mental health, chronic fatigue, and blood clots and/or circulatory system problems. Many of these long-term effects and impacts are still unknown.

As for specific examples, although the mortality rate of SARS-CoV2 is relatively low, its high infectivity causes a large part of the population to be infected simultaneously, saturating and collapsing health systems, thereby causing inadequate health care. disease and therefore increasing deaths caused by it. In addition to this, the saturation of health systems causes the neglect of other ailments, a situation that increases their mortality. Given this emergency situation, the search for pharmacological treatments to treat COVID 19, a disease caused by SARS-CoV2, has been a priority.

To combat the COVID-19 disease, the use of known drugs has been resorted to. Drugs such as chloroquine, hydroxychloroquine, azithromycin, remdesivir, lopinavir-ritonavir, favipiravir, IL-6 pathway inhibitors, ivermectin, corticosteroids, convalescent plasma, heparin, vitamin C, among others, have been tested as treatment. Although cases have been reported in which these drugs have been shown to help treat the disease, none of them have been found to be effective so far.

Background in the technical field

The root of Glycyrrhiza uralensis contains numerous compounds with pharmacological activity, including flavonoids, phenols with isoprenoid substitutions, isoflavonoids, saponins, and various volatile compounds. Among these compounds, the triterpene saponins, mainly glycyrrhizic acid (AG) and 18-b glycyrrhetinic acid (EN), present in licorice in the form of a mixture of their potassium and calcium salts, are of special interest. Glycyrrhizic acid (AG) and 18-b-glycyrrhetinic acid (EN) are pentacyclic triterpenoids of the olean type. These two have been identified as one of the main bioactive compounds of the Glycyrrhiza uralensis extract. Various pharmacological properties have been attributed to these.

Different studies point to the use of pentacyclic triterpenoids as an alternative in the prevention, treatment and post-treatment of infectious diseases of the respiratory system, due to their different physicochemical and pharmacological characteristics. J. Cinatl, et al, reported GA in 2003 as an inhibitor of SARS-CoV, preventing the replication of the virus in culture cells.

Harald Murck (MAY/28/2020) mentions that AG can function as a steric blocker of the ACE2 protein, which is the point of entry of SARS-CoV2 into the cell, it inhibits the expression of the TMPRSS2 protein, important for viral infection and in general that AG and its derivatives have antiviral properties.

Luo Pan, et al, (APR/29/2020) describes the pharmacological perspectives of AG, highlighting its role as an immunoregulator of cytokines, as a promoter of interferons with antiviral activity and as an inhibitor of the protein thrombin, important in the generation of clots that are seen in disease.

US5128149A SHANBROM (07/JUL/1992), describes a treatment of mammalian cells and biological fluids with compounds referred to herein as glycyrrhizic compounds [...] to inactivate viruses and to improve containers for providing such treatment. Additionally it relates to the treatment of whole blood to inactivate or destroy infectious viruses found in animal fluids and cells, such as cytomegalovirus which is responsible for aggravating infections in recipients of blood transfusions.

Document US2011052727A1 POLANSKY (03/MAR/2011), describes methods to prevent or treat an infection by an influenza virus. In a preferred embodiment, the methods include administering to a subject an effective dose of a nutritional supplement, wherein one of the components of the supplement is glycyrrhizic acid. Given this background, there are several sources that indicate the use of GA for the treatment, prevention and post-treatment of viral diseases. One of the problems with AG is the rapid metabolism it undergoes when administered to humans by oral or injected means. Due to this rapid metabolism, it has been suggested that oral or injected administration of FA may not achieve the local concentrations necessary to have a therapeutic effect. Furthermore, the concentrations necessary to observe an antiviral therapeutic effect are too high to be considered a completely useful drug. Other reported problems are irritability and toxicity when AG or EN are applied separately in relatively high concentrations, as will be seen later.

There are references where it is pointed out that a critical step in the infection is when the virus enters human host cells, which is enabled by the interaction between the SARS-CoV-2 Spike (S) protein on the surface of the viral particle and angiotensin converting enzyme 2 (ACE2) on the surface of human cells.

The present invention overcomes these limitations, providing pharmaceutical compositions adapted to be administered to a subject that comprise a synergistic combination of at least two pentacyclic triterpenoids, mainly by the inhaled route.

The invention is characterized by using optimal proportions with more than one pentacyclic triterpenoid in combination with pharmaceutically acceptable excipients, to block the interaction between the Spike(S) protein and ACE2 as a therapeutic target for the preventive treatment of lung diseases, reduce irritability and toxicity of individual components and prolong the systemic exposure of pentacyclic triterpenoids by the synergy between its components. Given the background and existing reports on the interaction of the drug glycyrrhizic acid (AG) with the ACE2 and Spike proteins, it was decided to test the effect of the combination of the latter and its derivative 18b-glycyrrhetinic acid (EN) in the inhibition of this interaction.

The resulting formulations are synergistic compositions whose proportions are designed to reduce toxicological effects, irritability and provide effective pharmacokinetics compared to the application of the same components individually.

The pharmaceutical forms according to the experimental developments are listed below: the preferred forms are solutions, suspensions and emulsions, as well as the powders and lyophilisates to prepare them, which are applied by means of nebulizations, vaporizations and sprays. Although there are various ways to classify them, in the present application they are indicated considering the route of administration.

- Oral administration: Solutions, suspensions and emulsions for oral administration, as well as solids that include tablets, coated tablets, modified release tablets, chewable tablets, capsules, soft capsules, hard capsules.

- Cutaneous administration: Solutions, suspensions, foams, pastes, spray powders (powders, solutions and suspensions administered by spray), gels, creams, ointments, patches and intradermal implants.

- Nasal administration: Drops formed by solutions, suspensions and emulsions. solution type spray, emulsions and suspensions. Nasal powders, administration gels and nasal ointments.

Injectables: Injectable solutions, suspensions and emulsions. Powders to prepare injectable solutions, suspensions and emulsions. Lyophilisates to prepare injectable solutions, suspensions and emulsions.

- Respiratory tract: Liquids such as solutions, suspensions and emulsions to be nebulized. Liquids such as solutions, suspensions and emulsions to be vaporized. Solids as powders and lyophilized to be administered through the oropharyngeal routes.

Description of the figures

Figure 1. Schematic of the COVID-19 Spike-ACE2 assay: Spike Complex - ACE2. If there is interaction between the ACE2 protein (II) and the SPIKE protein (I), the anti-SPIKE antibody (IV) coupled to the HRP enzyme (III) will bind to the spike protein (I). This complex will generate a color change in the ELISA assay (Plate V) that can be quantified.

Figure 2. Schematic of the COVID-19 Spike-ACE2 assay: SPIKE-ACE2 complex inhibition. In the presence of an inhibitory molecule (VI) such as glycyrrhizinic acid or enoxolone, the interaction between the SPIKE protein (I) and the ACE2 protein (II) is interrupted.

Figure 3. Scheme of the COVID-19 Spike-ACE2 assay: In the presence of an inhibitor (VI) there is no formation of the SPIKE-ACE2 complex, and therefore there is no coupling of the conjugated antibody to the HRP enzyme (III), for so there is no measurable color change in the ELISA test (Plate V). Figure 4. Graph of the ELISA assay for the quantification of the inhibition (or percentage of activity) of the ACE2 protein with the SPIKE protein.

Figure 5. Results of the inhibition of the formation of the ACE2-Spike complex, derived from the exposure of the ACE2 protein to the drugs AG, EN and the combination of both.

Figure 6. Images of histopathological sections of murine respiratory tract tissue with application of compositions with AG, EN and AG-EN combination.

Figure 7. Standardized table for the evaluation of toxicity and irritability of murine respiratory tract tissue with the application of compositions with AG, EN and a combination of AG-EN.

Figures 8 and 9. Pharmacokinetic graphs of EN in suspension, corresponding to 24 and 96 hours.

Figures 10 and 11. Pharmacokinetic graphs of EN in solution, corresponding to 24 and 96 hours.

Figure 12. Comparative graph of EN pharmacokinetics in suspension vs. solution, corresponding to 24 hours.

Figures 13 and 14. AG pharmacokinetic graphs corresponding to 24 and 96 hours

Figures 15 and 16. Pharmacokinetic graphs for the combination 30 mg AG and 2 mg EN (ratio 15:1) (037COV21), corresponding to 24 and 96 hours. Figure 17. Graph of the pharmacokinetic behavior of the AG-EN combination formulation (037COV21) against the individual administration of the drugs (038COV21 and 039COV21).

Figures 18 and 19. Pharmacokinetic graphs for the combination 60 mg AG and 3 mg EN (ratio 20:1) (036COV21), corresponding to 24 and 96 hours.

Figure 20. Pharmacokinetic graph for different formulations corresponding to 24 hours.

Figure 21. Pharmacokinetic graph for different formulations corresponding to 24 hours.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to pharmaceutical compositions that are adapted to be administered nasally, cutaneously, orally, by injection or inhalation for use in the prevention, treatment and post-treatment of respiratory tract diseases. These compositions are composed of synergistic combinations of pentacyclic triterpenoids together with suitable and pharmaceutically acceptable excipients for the different routes of administration. The present pharmaceutical compositions are based on the unexpected synergistic effect of some combinations of specific proportions of pentacyclic triterpenoids. The compositions described here are especially effective in the treatment of viral infections, especially those caused by contagion via the interaction of the virus with the angiotensin-converting enzyme 2 (ACE2).

The various compositions of the present invention contain at least two pentacyclic triterpenoids. specifically it refers to glycyrrhizic triterpenoids as the group of molecules selected from the group comprising glycyrrhizic acid (also designated as AG), its aglycone, 18b-glycyrrhetinic acid (also designated as EN) and derivatives thereof, in the form of acids, salts , esters and other derivatives. Regarding the group of glycyrrhizic triterpenoids of the present formulation, the combination of glycyrrhizic acid (AG) and 18b-glycyrrhetinic acid (EN) is preferably used due to its inhibitory effect on the interaction pathway between the Spike protein (S) of the virus with the angiotensin 2 Converting Enzyme,

(ACE2).

The experiments carried out consisted of testing the ELISA RayBio COVID-19 Spike-ACE2 binding assay kit (Catalog Number: CoV-SACE2-l), described as an assay for the detection of antibodies and drugs against COVID-19 disease. The purpose of this assay is to quantify the formation of the ACE2-Spike complex and how it is affected by the presence of a drug.

The assay is described by Figures 1 to 3 showing the scheme of the COVID-19 Spike-ACE2 assay, in Figure 1, the Spike-ACE2 complex that forms when there is no inhibitor is exposed; Figure 2 shows how the formation of the Spike-ACE2 complex is blocked in the presence of an inhibitory molecule (VI); whereas, Figure 3 shows that, if the drug of interest affects the formation of the Spike-ACE2 complex, the complex of interactions will not be formed as shown in the figure, and therefore there will be no quantifiable color change. On the contrary, if the drug does not inhibit the interaction of the complex, a strong staining signal will be obtained.

Figure 4 shows the behavior of the tested composition in ELISA assays for the quantification of the inhibition (Percentage of activity) of the ACE2 protein with the SPIKE protein at different concentrations of glycyrrhizic acid (AG) alone, 18b-glycyrrhetinic acid (EN) alone, a blank (P) and the combination of AG-EN in proportions within the range of 1:3 to 1:25, preferably within the range of 1:5 to 1:20. The combination of triterpenoids turns out to be more effective than the use of the active ingredients separately. This derives in the technical advantage that the use of a lower concentration of both drugs results in obtaining an improvement related to the therapeutic effect.

Figure 5 shows the results of the inhibition of the formation of the ACE2-Spike complex, derived from the exposure of the ACE2 protein to the drugs AG, EN and the combination of both. It is important to note that the X-axis shows two different scales, corresponding to the concentration in micromoles (mM) of compounds AG and EN. It is important to appreciate that the combination of AG-EN in proportions within the range of 1:3 to 1:25, preferably within the range of 1:5 to 1:20, turns out to be obviously more effective than the use of the active principles by separate.

The graph in Figure 5 shows the different concentrations of both drugs (glycyrrhizic acid (AG) and 18b-glycyrrhetinic acid (EN)) and the percentage of binding of the Spike-ACE2 complex that is detected at these concentrations. The graph shows the 3 formulations with the active ingredient. The dotted line with squares (F-EN) shows the behavior of the formulation with EN, the continuous line with circles (F-AG) the formulation with AG and the dashed line with triangles shows the formulation with both compounds. In the lower part, the concentrations in units of micromoles (mM) of both drugs are shown, valid for the three formulations (AG concentrations valid for F-AG and F-EN, EN concentrations valid for for F-EN and F-AG-EN). It is important to highlight that the concentration of AG with respect to that of EN is in a ratio of ~5.7: 1, (when there is approximately 88% inhibition) in molar terms, while this ratio is 10 to 1 (AG with respect to enoxolone ( EN)) in terms of mass, achieving significant inhibition at lower applied doses.

The formulations described here, in addition to the glycyrrhizinous triterpenoids, contain pharmaceutically acceptable excipients that provide the formulations with pharmacological properties necessary to be administered in humans. Specifically, the present invention contains components such as ionic solubilizers, surfactants, non-ionic solubilizers, solvents, pH regulators, osmotic regulators, chelating agents, antioxidants and preservatives.

In the present invention, ionic solubilizers are defined as the group selected from propylene glycol, glycerin/PEG, poloxamer, polyoxylglyceride derivatives (Labrasol), glyceryl isostearate / glyceryl monostearate, glycerol derivatives, polyethylene glycol 66012-hydroxystearate, castor oil and its derivatives. The invention defines surfactants including, but not limited to polysorbate, sorbitan monostearate, sorbitol esters, polysorbate 20,60,80 (derivatives of polyoxyethylene sorbitan fatty acid esters). The invention defines nonionic solubilizers as the group selected from diethylene glycol monoethyl ether; as well as the solvents to the group selected from ethanol and water.

Optionally, the formulations can be subjected to pH adjustment with HC1, NaOH or any salt and its mixture that form an indicated buffer. To adjust the pH between formulations they can also include an osmolarity adjuster, such as NaCl, dextrose, mannitol and sorbitol. The formulations may also contain preservative elements such as benzalkonium chloride, ethanol, propylene glycol, benzyl alcohol, chlorobutanol, paraben derivatives. The compositions may include chelating agents for example EDTA and derivatives, citric acid and derivatives, oxalic acid and derivatives, ascorbic acid and derivatives. The compositions may include components to adjust viscosity, for example, poloxamer in its different degrees of polymerization, microcrystalline cellulose and derivatives; as well as the inclusion of antioxidants such as L-cysteine, butylhydroxytoluene, butylhydroxyanisole.

In a preferred embodiment, the formulation uses a combination of AG and EN, in concentrations for AG from 0.1% to 20% (m/m) and from 0.01% to 4% m/m for EN, preferably AG from 0.1% to 10% (m/m) and from 0.01% to 2% m/m for EN. In this modality, the formulation uses propylene glycol as ionic solubilizer in concentrations from 70% to 80% (m/m), polysorbate as surfactant in concentrations from 0.1 to 7% (m/m), diethylene glycol monoethyl ether as non-ionic solubilizer in concentrations from 1% to 6% (m/m) and water as solvent in a range from 10% to 20% (m/m). In this mode, the pH is adjusted to values between 4.0 and 9.0, preferably between 4.5 and 6.5, using one or more of the elements mentioned above.

Having stated the above, the pharmaceutical composition now disclosed comprises: a synergistic combination of at least two pentacyclic triterpenoids in a ratio ranging from 1:3 to 1:25 for use in the treatment of viral infections.

In other embodiments, the viral infections are viral respiratory infections. In other embodiments, the composition comprises at least one pharmaceutically acceptable additive, where the pentacyclic triterpenoids are glycyrrhizinic acid and 18b-glycyrrhetinic acid, and are found in a ratio preferably in the range of 1:5 to 1:20.

In other embodiments, the pharmaceutical composition has a glycyrrhizinic acid content of 0.1% to 20% and a 18b-glycyrrhetinic acid content of 0.01 to 4%, preferably AG of 0.1% to 10% (m/m) and 0.01 % to 2% m/m for EN.

In other embodiments, the pharmaceutical composition has a content of the synergistic combination within the composition, which is in the range of greater than 0.0% to 30%, preferably in the range of 0.01% to 10%.

In other embodiments, pharmaceutically acceptable additives comprise ionic and nonionic solubilizers, surfactants, and solvents; where the ionic solubilizers are selected from the group comprising: propylene glycol, glycerin / PEG, poloxamer, polyoxylglyceride derivatives (Labrasol), glyceryl isostearate / glyceryl monostearate, glycerol derivatives, polyethylene glycol 66012-hydroxystearate, castor oil and its derivatives , and are found in a concentration of 70% to 80% m/m.

In other embodiments, the pharmaceutical composition comprises surfactants that are selected from the group comprising polysorbate, sorbitan monostearate, sorbitol esters, polysorbate 20,60,80 and are found in concentrations from 0.1% to 7%.

In other embodiments, the pharmaceutical composition comprises diethylene glycol monoethyl ether as a non-ionic solubilizer in a concentration from 1% to 6% and as solvent water, in the range from 10% to 20%.

Examples: In tests carried out, the following formulations were tested:

• Formulation 1: Nebulizable solution AG 2.8%

• Formulation 2: Nebulizable solution EN 0.28%

• Formulation 3: Nebulizable solution AG-EN 2.8% - 0.28%.

• Formulation 4: Excipients or white nebulizable solution.

The respective formulations, in addition to containing the active ingredients (AG AND EN), contain:

• Propylene glycol from 70 to 80%; preferably 73 to 77% m/m.

• Polysorbate from 0.1% to 7%; preferably 4 to 6% m/m.

• Diethylene glycol monoethyl ether from 1% to 6%; preferably 3 to 5% m/m.

• Water, balance at 100% m/m.

The different formulations were tested in 4 different dilutions, starting from the original concentrations and making 3 additional 1:1 dilutions. The concentrations used in the study are shown in the following table:

Table 1.

Table 2.

Tables 1 and 2 show comparative formulations containing AG and EN individually and specifically in formulation 3, a 1:10 ratio between EN-AG is indicated for the inhibition assay of the SPIKE-ACE2 protein.

Results: As a result of the experiment, the following values were obtained in terms of percentage inhibition of the Spike-ACE2 complex.

Table 3.

One of the problems with AG is the rapid metabolism it undergoes when administered to humans by oral or injected means. Given this rapid metabolism, it has been suggested that oral or injected administration of FA may not reach the local concentrations necessary to have a therapeutic effect. Furthermore, the concentrations necessary to observe an antiviral therapeutic effect are too high to be considered a drug. The present application describes, therefore, the characteristics of the invention, evaluating the toxicological effects and local irritability in the use of the synergistic composition by the inhaled route in murine.

Tables 4 and 5 show comparative formulations containing AG and EN individually and AG-EN for irritability and toxicity tests in murine.

Table 4.

Table 5.

These formulations were administered to groups of murine for 10 minutes a day for 14 consecutive days. Each group was given only one type of dose for the entire duration of treatment. At the end of the treatment, the animals were sacrificed and their respiratory tract tissue was recovered. For each group, lung tissue and trachea were recovered from one murine. Said tissue was subjected to a histopathological analysis, where the tissue damage of said samples was evaluated as follows:

"The alterations observed between the different groups are classified in Table 4 with a subjective score of 0 to 5, where 0 is absence of injury and 5 corresponds to severe injury." See figures 6 and 7.

The cuts shown in Figure 6 were examined and assigned a damage rating based on their criteria. The evaluation mentioned is found in table 6:

Table 6. In Table 6, the last row indicates the total damage and injury rating by group. These results were normalized by subtracting the control score (control = 9.5). The normalized data are presented in Figure 7.

The results of figure 6 show a series of patterns with respect to the different compositions:

• The composition with AG indicates the generation of additional lesions to the basal lesions, which increases according to the amount of drug administered.

• The composition with EN indicates a decrease in basal lesions when used at low concentrations. At higher concentrations of this compound, this effect is lost and it begins to cause additional injuries.

• The combination of both drugs indicates that it has a joint beneficial effect greater than what would be expected from the effect of both individually.

. For the case of the combined low AG-EN composition (4 mg/mL AG + 0.4 mg/mL EN) the expected sum of the individual effects is -3 (-3.5 + 0.5) and the observed effect is -4.

. For the case of the high AG-EN composition (10 mg/mL AG + 1 mg/mL EN) the sum of the expected effects is 3.5 (3 + 0.5), while the observed effect is -3.5

The results suggest that there is an unexpected advantage by using a high AG-EN composition (10 mg/mL AG + 1 mg/mL EN). On the one hand, it is suggested that the lesions expected by the individual use of its components (value of 3.5) are counteracted by the combination of these, and it is even observed that this combination synergistically reduces pre-existing lesions given the value observed (value of -3.5). In addition, these results suggest that a higher dose can be used, which would mean observing a more powerful therapeutic effect. Alternative tests were carried out to verify the synergistic behavior of the compositions of interest. Therefore, the results of pharmacokinetic studies are described and disclosed. The study was rooted in the application of different versions of the pharmaceutical composition that is now required to healthy volunteers by the inhaled administration route, by means of a nebulizer device. The volunteers were given the drug every 24 hours for three days. These same blood samples were taken at different time points. These samples were analyzed to quantify the contents of glycyrrhizinic acid (AG) and Enoxolone (EN) by means of the mass-coupled HPLC methodology. The study consisted of the application of the following formulations to healthy volunteer individuals:

Table 7.

Table 8. Comparative formulations containing AG and EN individually and AG-EN for pharmacokinetic testing are shown in Tables 7-11.

For the pharmacokinetic study, each individual was given 1 mL of one of the compositions, diluting it with 4 mL of saline solution. The resulting mixture was supplied through a Nebucore model P-103 nebulizer device, for a period of approximately 20 minutes or until the solution was completely nebulized (whichever occurred first). Subjects were given a second and third dose of the drug at 24 and 48 hours, respectively.

Blood samples of 5 mL were taken from each of the individuals at 30 minutes and at 1, 2, 3, 6, 12, 24, 48, 72 and 96 hours. The samples were used to determine the concentration of the drugs AG and EN by means of the HPLC methodology coupled to masses. In all cases, both drugs were quantified. The results are shown below.

The following tables show the pharmacokinetic results (area under the curve (AUC)) of the two drugs analyzed (AG and EN). The area under the curve (AUC) is a parameter related to the amount of drug to which the body has been exposed. Results were obtained by analyzing plasma concentrations at 24 hours. For the calculation of the ABC, the "PK" package was used within the R programming language. For the calculation of these, a single-compartment model was assumed.

ABC Enoxolone 24 hours

Table 9. ABC AG 24 hours

Table 10. Figures 8 to 21 show the graphs corresponding to the changes in concentration in the blood samples over time. Two temporalities are shown for respective formulations: 24 and 96 hours. The analyzes and conclusions mentioned correspond to the graphs at 24 hours. This is so, since most of the samples collected (30 minutes, 1, 2, 3, 6, 12 and 24 hours) characterize the pharmacokinetic behavior of the formulations from the first intake (0 hours), until before the second administration (24 hours). Figures 8, 9, 10 and 11 show, respectively, the pharmacokinetic graphs of EN in suspension 040COV21 (fig. 8 and 9) and EN in solution 039COV21 (fig. 10 and 11) corresponding to 24 and 96 hours. Figure 12 shows a comparative graph of the pharmacokinetics of EN in suspension vs. EN in solution corresponding to 24 hours. The effect observed in the graph of figure 12, Enoxolone suspension for nebulization (includes undissolved particles), shows smaller areas under the curve with respect to Enoxolone Solution for nebulization (completely dissolved particles), a result that delimits and confirms the virtues between the selection of a pharmaceutical form in the absorption of the drug), and the effect produced by the reduction of the particle size of the drugs that potentiates the permeation either by diffusion, carry or ion exchange; Additionally, it is indicated that the smaller the particle size and moderate permeability, the greater its absorption; another point to consider in the selection of the Pharmaceutical Form (FF) and the route of administration where not only the biopharmaceutical and physicochemical properties influence the administration of the product, but also the selection of the inputs or excipients will modulate the delivery, stability , reproducibility and administration of the drug.

For the specific case of glycyrrhizic acid (AG), it can be mentioned that its absorption by the inhalation route is dependent on the pharmaceutical form, since although AG is soluble in water, it has a reduced permeability, therefore, the excipients and manufacturing process will limit its absorption. Regarding the results obtained, we can observe in the graphs of figures 13 and 14 two absorption curves, one close to 24 hours after the application of the product and a second at 96 hours, observing a representative effect of the pharmaceutical form of release. modified, an effect that we can correlate to said pharmaceutical form and to the route of administration where it is estimated that the airways remain impregnated with AG for more than 24 hours; additionally, an initial maximum is observed at 60 min, characteristic of the drug when it is administered orally (gastrointestinal absorption) and that is where AG is biotransformed to EN, a metabolite that will undergo a second metabolism in the liver to later be eliminated of the organism. On the other hand, the data obtained show that the elimination of FA is prolonged for a longer time when it is administered by the inhaled route, showing a bicompartmental behavior that is also observed when the drug is administered by the injectable route and being very different from the injectable route. oral (two ascending plateaus) where its Elimination starts after 6 o'clock. On the other hand, regarding the advantages of the developed formulation, a solution with low viscosity was obtained, which gives an advantage over other pharmaceutical forms with similar concentrations, since products with high concentrations tend to form gels due to the effect of glycyrrhizinic acid, AG.

In graphs 13 and 14, there is a presence of EN, this is because, as mentioned above, AG is biotransformed (hydrolyzed) to EN to be metabolized in the liver and subsequently eliminated. On the other hand, the concentration obtained by inhalation is much lower than that obtained orally (200.3 ng/mL when 75 mg of AG are administered), which is an indicator that it is a biotransformation that occurs in the gastrointestinal tract; Therefore, when FA is absorbed without passing through the gastrointestinal tract, its metabolism to EN is reduced, metabolizing only the fraction that manages to pass through this pathway.

Due to the above, it can be pointed out that the administration of GA by air route (in nebulized solution), provides an initial surface effect in the upper airways, exerting its antimicrobial and antiviral effect, to subsequently produce a prolonged systemic effect (greater than 24h), with reduced biotransformation towards EN.

In figures 15 and 16, corresponding to the 037COV21 formulation, the results in the initial times show clear differences with respect to the administration of AG and EN individually, where their administration in the form of a mixture has effects on their absorption, distribution and metabolization, obtaining a higher C max compared to formulation _038COV21 (with 60mg AG in the absence of EN). Concerning the results obtained after 24 hours, no further significant differences were observed.

On the other hand, the absorption of EN shows a synergistic effect obtained by the effect of the 037COV21 mixture and whose area under the curve is greater than that obtained in the formulation 039COV21 (EN 3mg) and 038COV21 (metabolization of AG) (1258.47 vs 664.50 and 253.41), but being slightly higher than that referenced orally (AG 75 mg). Additionally, no significant differences were observed regarding the behavior of the 037COV21, 038COV21 and 039COV21 formulations after 24 hours. Figure 17 shows a comparison of the AUC of the combination formulation (037COV21) against the individual administration of the drugs (038COV21 and 039COV21); As can be seen in the AUC values (1258.47 vs. 664.50 and 253.41 for EN) (9372.09 vs. 2297.631 for AG), the combination formulations show a greater systemic exposure of both drugs compared to that expected by the individual administration of the same. From the previous results, it can be highlighted that the administration of these drugs by the airway focuses the target organ and that, in addition, systemic effects similar to the administration of AG by the oral route are obtained.

As shown in figures 18 and 19, the formulations that include a combination of AG and EN show values of area under the curve higher than the values of the individual administration of these drugs. Figure 20 shows a synergistic effect when comparing these combinations with each other. It is observed that changes in the proportion of the active compounds (20:1 in the 036COV21 sample vs 15:1 corresponding to the 037COV21 sample) have an impact on the behavior of the pharmacokinetic curves, specifically in their respective AUC (3594.57 for 036COV21 ) vs (1258.47 for 037COV21) corresponding to EN and (6510.27 for 036COV21) vs (9372.09 for 037COV21) corresponding to AG. Said changes cannot be explained only by changes in the concentrations of the pharmacokinetic formulations, since, as observed, the AUC values of the formulations are not proportional to the concentrations used of the active compounds.

Although both formulations show technical advantages compared to the individual administration of AG and EN, the preference for a combined pharmaceutical form (036COV21 or 037COV21) depends on the drug to be potentiated in terms of systemic exposure. For example, if it is desired to potentiate the exposure to AG, it is preferably suggested to use the formulation 037COV21; while, if you want to potentiate EN exposure, it is suggested to preferably use the 036COV21 formulation.

Table 11.

These data as a whole show the technical advantage of AG and EN pharmaceutical formulations compared to the individual administration of both drugs. In addition to the increase in the AUC of both components in the combined formulations, the results show that changes in the proportions of both drugs modulate their systemic exposure, independently of the concentration used in the formulation. The usefulness of this modulation, the preferred proportions and concentrations are based on the therapeutic interests of the use of these formulations. Analysis

The results obtained for both analytes within the 036COV21 formulation (AG ratio 20:1 EN) delimit the behavior of the modified release pharmaceutical form, for which two maximums are obtained before 24 hours, it is the same effect observed in the formulation 038COV21 (AG) but obtaining higher plasma concentrations due to the effect of EN on AG as previously mentioned. Regarding EN, a lower initial concentration close to 150 ng/mL is observed, but it is lower than that obtained in the 037COV21 formulation (AG ratio 15:1 EN), an effect that limits its absorption, but increases its residence time, that is, the therapeutic effects of EN, such as antiviral action and antimicrobial effect can be enhanced due to the increase in residence time; on the other hand, the second maximum before 24 hours presents a plasma concentration close to 250 ng/mL, a maximum not observed in the study formulations and higher than that obtained orally in administrations of AG 75 mg.

Additionally, it is observed that EN increases its half-life and AUC with respect to the 037COV21 formulation, defining a bicompartmental absorption, that is, the analyte is absorbed and distributed to the organs to exert its therapeutic effect or be metabolized (liver), but not being retained or metabolized, it is recirculated to the circulatory system (CS), to restart its metabolism again, which leads to the prolongation of the therapeutic effect; This effect is usually attributed to the plasma concentration of the analytes, that is, when the plasma concentration exceeds the defined amount of enzymes that promote their metabolism, the liver recirculates the intact NE to the SC to subsequently restart its metabolism and elimination from the body.

Due to the above, it can be pointed out that the proportion defined in the 036COV21 formulation conditions its absorption, by enhancing the permeation of the AG drug and by limiting the absorption of EN.

The plasma concentration obtained delimits its elimination in a dose-dependent manner, which leads to an increase in the residence time of the drugs and thus, to a potentiation of their systemic therapeutic effects.

Claims

CLAIMS Having described the invention as above, the contents of the following claims are claimed as property of the owner:

1.- A pharmaceutical composition comprising a synergistic combination of at least two pentacyclic triterpenoids in a ratio ranging from 1:3 to 1:25 for use in the treatment of viral infections.

2. The pharmaceutical composition for use according to the preceding claim, wherein the viral infection is a respiratory viral infection.

3. The pharmaceutical composition according to claim 1, characterized in that it comprises at least one pharmaceutically acceptable additive.

4. The pharmaceutical composition according to claim 1, characterized in that the pentacyclic triterpenoids are glycyrrhizinic acid and 18b-glycyrrhetinic acid.

5. The pharmaceutical composition according to any of the preceding claims, characterized in that it comprises a synergistic combination of at least two pentacyclic triterpenoids in a ratio preferably in the range of 1:5 to 1:20.

6. The pharmaceutical composition according to any of the preceding claims, characterized in that the content of glycyrrhizinic acid is from 0.1% to 20% and the content of 18b-glycyrrhetinic acid is from 0.01 to 4%.

7. The pharmaceutical composition according to any of the preceding claims, characterized in that the content of the synergistic combination is in the range of greater than 0.0% to 30%, preferably in the range of 0.01% to 10%.

8. The pharmaceutical composition according to claim 3, characterized in that the pharmaceutically acceptable additives comprise ionic and non-ionic solubilizers, surfactants and solvents.

9. The pharmaceutical composition according to claim 8, characterized in that the ionic solubilizers are selected from the group comprising: propylene glycol, glycerin / PEG, poloxamer, polyoxylglyceride derivatives (Labrasol), glyceryl isostearate / glyceryl monostearate, glycerol derivatives , polyethylene glycol 660 12-hydroxystearate, castor oil and its derivatives.

10.- The pharmaceutical composition according to claim 9, where the ionic solubilizers are in a concentration of 70% to 80% m/m.

11. The pharmaceutical composition according to claim 8, wherein the surfactants are selected from the group comprising polysorbate, sorbitan monostearate, sorbitol esters, polysorbate 20,60,80.

12.- The pharmaceutical composition according to claim 11, where the surfactants mentioned in the claim are found in concentrations from 0.1% to

7%.

13. The pharmaceutical composition according to claim 8, where diethylene glycol monoethyl ether is used as non-ionic solubilizer.

14.- The pharmaceutical composition according to claim 13, wherein the nonionic solubilizer is in a concentration of 1% to 6%.

15. The pharmaceutical composition according to claim 8, where water is used as a diluent, in the range of 10% to 20%.